Thousands of terpenes and terpenoid derivatives found throughout Nature are involved in diverse biosynthetic and metabolic pathways such as cholesterol biosynthesis in humans and paclitaxel (Taxol) synthesis in the Pacific yew. Notably, many terpenoids have been used as medicinal agents since times of antiquity due to their analgesic, antibiotic, and antifungal properties. In spite of the universal importance of this family of natural products for human health, it is remarkable that the three-dimensional structures of terpenoid cyclases have only been reported relatively recently. Terpenoid cyclases (a.k.a. synthases) catalyze the specific cyclization of a common allylic pyrophosphate substrate, such as farnesyl diphosphate, into one of hundreds of possible products. The terpenoid cyclase plays a critical role as a template in "channeling" the precise substrate and intermediate conformations leading to the formation of one exclusive product. Thus, the terpenoid cyclases comprise an exciting class of biosynthetic enzymes from both the biological and the chemical perspectives. In the current funding period, we have determined the first X-ray crystal structure of a monoterpene cyclase, (+)- bornyl diphosphate synthase;we have determined the fifth crystal structure of a sesquiterpene cyclase, aristolochene synthase from A.terreus;and we have established the structural basis for aberrant product formation by site-specific variants of trichodiene synthase. We aim to build upon this outstanding foundation in the next funding period by dissecting detailed structure-biosynthetic diversity relationships in trichodiene synthase. Specific!ally, we will study site-specific variants with altered metal binding properties, and we will also study variants engineered to generate alternative products. We will also study the structural basis for the evolution and fidelity of aristolochene synthase from A. terreus and P. roqueforti. In order to broaden our knowledge of structure-function relationships in the greater family of terpenoid cyclases, we will also study the diterpene cyclase taxadiene synthase. Finally, we will determine the X-ray crystal structure of sterol methyltransferase, a potential drug target for the treatment of fungal infections. Now that we have established a solid foundation in the study of biosynthetic enzymes that generate cyclic terpene products, we will now study an enzyme that utilizes a cyclic terpene substrate in a novel chemical reaction that further diversifies the biosynthetic array of cyclic terpene natural products.